PEG conjugates of NK4

ABSTRACT

The present invention provides a conjugate consisting essentially of a NK4 molecule and a polyethylene glycol group having a molecular weight of from about 20 to about 40 kDa. The invention also provides a composition in which the monoPEGylated conjugates comprise at least 90% of the total of pegylated NK4 molecules and unpegylated NK4 molecules in the composition. Also provided is a composition in which the monoPEGylated conjugates comprise conjugates in which the PEG groups are attached to groups randomly selected from the lysine side chains of NK4 molecules and the N-terminal amino groups of NK4 molecules. A method for the treatment of cancer by administering 1 to 30 mg monoPEGylated NK4 per kg per day is further provided.

BACKGROUND OF THE INVENTION

[0001] Hepatocyte growth factor (HGF/SF) is a polypeptide identified andpurified by Nakamura, T., et al., Biochem. Biophys. Res. Commun. 22(1984) 1450-1459. It was further found that hepatocyte growth factor isidentical to scatter factor (SF), Weidner, K. M., et al., Proc. Natl.Acad. Sci. USA 88 (1991) 7001-7005. HGF is a glycoprotein with amolecular weight of about 100 kDa involved in the development of anumber of cellular phenotypes including proliferation, mitogenesis,formation of branching tubules and, in the case of tumor cells, invasionand metastasis. For a status review, see Stuart, K. A., et al.,International Journal of Experimental Pathology 81 (2000) 17-30. Bothrat HGF and human HGF have been sequenced and cloned (Miyazawa, K. etal., Biochem. Biophys. Res. Comm. 163 (1989) 967-973; Nakamura, T., etal., Nature 342 (1989) 440-443; Seki, T., et al., Biochem. and Biophys.Res. Comm. 172 (1990) 321-327; Tashiro, K., et al., Proc. Natl. Acad.Sci. USA 87 (1990) 3200-3204; Okajima, A., et al., Eur. J. Biochem. 193(1990) 375-381). The pharmacokinetics and pharmacological effects of anHGF lacking the first five N-terminal amino acids (dHGF) wereinvestigated by Uematsu, Y., et al., J. Pharm. Sciences 88 (1999)131-135. It was found that the serum concentration of dHGF decreasedrapidly and therefore infusion would be preferred against bolusinjection as administration route.

[0002] U.S. Pat. No. 5,977,310 describes PEG-modified HGF. SuchPEG-modified HGF has a prolonged clearance in vivo and has the samephysiological activity as HGF. However, according to U.S. Pat. No.5,977,310, it is only possible to prolong the half life of HGF from 59.2minutes to 76.7 minutes or 95.6 minutes, respectively (see Example 5 ofU.S. Pat. No. 5,977,310). It is further suggested in this patent thatthe molar amount of the PEG reagent may be selected from the range offrom 5 to 100 times of the molar weight of HGF. In the case of modifyingan amino group of lysine or the N-terminus of protein, a preferred molarrange of the PEG reagent is of from 10 to 25 times of the molar weightof HGF. The molecular weight of the attached PEG chain was about 10 kDa.Methods for the synthesis of conjugates consisting of PEG andpolypeptides such as HGF are also described in WO 94/13322. Theseconjugates are linked together at predefined positions as randomconjugation leads, according to the authors, to the introduction ofpolymeric moieties into domains of the molecule that mediate thetherapeutically or diagnostically desirable activities. Consequently,the molecules may aquire a prolonged half-life in vivo and, in the caseof heterologous proteins, reduced immunogenicity, but at the expense ofa significant or complete loss of the desired biological activities(see, e.g., Kitamura, K., et al., Cancer Res. 51 (1991) 4310-4315 andMaiti, P. K., et al., Int. J. Cancer Suppl. 3 (1988) 17-22). PEGylatedIFN-α shows, for example, only 7% of the potency compared tonon-PEGylated IFN-α (Bailon, P., Bioconjugate Chem. 12 (2001) 195-202).

[0003] It was further found that an HGF/SF fragment, termed NK4,consisting of the N-terminal hairpin domain and the four kringle domainsof HGF/SF, has pharmacological properties that are completely differentfrom those of HGF/SF, and is an antagonist to the influence of HGF/SF onthe motility and the invasion of colon cancer cells, and is, inaddition, an angiogenesis inhibitor that suppresses tumor growth andmetastasis (WO 93/23541; Parr, C., et al., Int. J. Cancer 85 (2000)563-570; Kuba, K., et al., Cancer Res. 60 (2000) 6737-6743; Date, K., etal., FEBS Letters 420 (1997) 1-6; Date, K., et al., Oncogene 17 (1989)3045-3054; Tomioka, D., et al., Cancer Res. 61 (2001) 7518-7524).

[0004] As emerges from Kuba, K., et al., Cancer Res. 60 (2000)6737-6743, in animal experiments, for detecting an effect of NK4 on lungmetastases, NK4 had to be infused continuously over a period of twoweeks.

[0005] It is known that the attachment of polymers to certainpolypeptides may increase the serum half life of such polypeptides. Thiswas found, for example, for PEGylated Interleukin-6 (EP 0 442 724) orInterleukin-2 (WO 90/07938) and erythropoietin (WO 01/02017). However,the attachment of polyethylene glycol and other polymers does notnecessarily lead to prolongation of their serum half lives. It is known,for example, that the conjugation of different polyethylene glycols toInterleukin-8, G-CSF and other interleukins results in the production ofmolecules with impaired properties (Mehvar, R., J. Pharm. Pharm. Sci. 3(1) (2000) 125-136). Thus, the outcome of a PEGylation of a polypeptideis highly unpredictable. Gaertner, H. F., and Offord, R. E.,Bioconjugate Chem. 7 (1996) 38-44 describes the site-specific attachmentof PEG to the amino terminus of proteins. Gaertner et al. state (asalready mentioned in WO 94/13222, see above) that PEGylation presents abig problem if the attachment sites cannot be precisely controlled, asthis might have important implications for protein stability andfunction.

[0006] Francis, G. E., et al., Int. J. Hematol. 68 (1998) 1-18 presentan overview of PEGylation of cytokines and other therapeutic proteins.Francis et al. state that with the majority of methods of PEGylation,substantial reduction of bioactivity has been reported (typically,20-95%). According to Francis et al., PEGylation of proteins is alwaysbased on trial and error and virtually all parameters of such aPEGylation can have a surprising and very profound effect on thefunctionality of the product. Tsutsumi, Y., et al., Thromb. Haemost. 77(1997) 168-173 describes the PEGylation of Interleukin-6. According toTsutsumi et al., about 54% of the lysine amino groups of IL-6 werecoupled with PEG with a molecular weight of 5 kDa per PEG group.Tsutsumi et al., in Proc. Natl. Acad. Sci. USA 97 (2000) 8548-8553,describe the chemical modification of an immunotoxin by PEG. As randomPEGylation was accompanied by a significant loss of specific cytotoxicactivity, Tsutsumi performs a site-specific PEGylation by using animmunotoxin mutant with one or two additional cysteins which are usedfor PEG coupling. Heinzerling, L., et al., Dermatol. 201 (2000) 154-157describes the coupling of PEG to Interferon-α with a molecular weight of5 kDa. Tsutsumi, Y., et al., in J. Pharmacol. Exp. Ther. 278 (1996)1006-1011, describe the PEG modification of TNF-α, whereby the molecularweight of the PEG groups used is again 5 kDa. As the PEGylated TNF-αapplied has a molecular weight of at least 84 kDa (by a molecular weightof 17 kDa of TNF-α there are at least 13 5-kDa PEG groups attached toTNF-α.

[0007] PEGylation of proteins and its pharmacological effects are alsoreviewed by Reddy, K. R., Ann. Pharmacotherapy 34 (2000) 915-923. Againit is stated that PEGylation of therapeutic proteins must be carefullyevaluated. Each protein is, according to Reddy et al., different,requires different optimization chemistry and therefore the influence ofPEGylation cannot be predicted.

SUMMARY OF THE INVENTION

[0008] The present invention provides a conjugate consisting of a NK4molecule and a polyethylene glycol group having a molecular weight offrom about 20 to about 40 kDa.

[0009] The present invention provides a composition comprisingconjugates of NK4 monoPEGylated with polyethylene glycol groups thathave a molecular weight of from about 20 to about 40 kDa, wherein theconjugates comprise conjugates in which the polyethylene glycol groupsare randomly attached to groups of the NK4 molecules selected from thelysine side chains of NK4 molecules and the N-terminal amino groups ofNK4 molecules.

[0010] The present invention also provides a composition comprisingconjugates of NK4 monoPEGylated with polyethylene glycol groups thathave a molecular weight of from about 20 to about 40 kDa, wherein themonoPEGylated conjugates comprise at least 90% of the total of pegylatedNK4 molecules and unpegylated NK4 molecules in the composition.

[0011] The present invention further provides a method for the treatmentof cancer wherein 1 to 30 mg of the monoPEGylated NK4 is administeredper kg per day to a patient in need of treatment.

BRIEF DESCRIPTION OF THE SEQUENCES

[0012] SEQ ID NO:1 shows the DNA and polypeptide sequence of NK4.

[0013] SEQ ID NO:2 shows the polypeptide sequence of NK4.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1 Inhibition of HGF-induced HUVEC proliferation by NK4, 20kDa-mono-PEG-NK4 and 30 kDa-mono-PEG-NK4.

[0015]FIG. 2 Inhibition of HGF-induced HUVEC proliferation by NK4 and 40kDa-mono-PEG-NK4.

[0016]FIG. 3 Inhibition of bFGF-induced HUVEC proliferation by NK4, 30kDa-mono-PEG-NK4 and 40 kDa-mono-PEG-NK4.

[0017]FIG. 4A Time-concentration curve of NK4, 30 kDa-monoPEG-NK4, 40kDa-mono-PEG-NK4 in plasma after i.v. administration.

[0018]FIG. 4B Time-concentration curve of NK4, 30 kDa-monoPEG-NK4, 40kDa-mono-PEG-NK4 in plasma after s.c. injection.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides NK4 conjugates consisting of NK4being covalently linked to one polyethylene glycol (PEG) group of fromabout 20 to about 40 kDa (monoPEGylated NK4), preferably via an ε-aminogroup of NK4 lysine or the N-terminal amino group. Most preferably, NK4is randomly PEGylated at one amino group out of the group consisting ofthe ε-amino groups of NK4 lysine and the N-terminal amino group.Surprisingly, it has been found that monoPEGylated NK4 according to theinvention has superior properties in regard to the therapeuticapplicability of NK4 or otherwise PEGylated NK4.

[0020] Human HGF is a disulfide-linked heterodimer, which can be cleavedin an α-subunit of 463 amino acids and a β-subunit of 234 amino acids,by cleavage between amino acids R494 and V495. The N-terminus of theα-chain is preceded by 31 amino acids started with a methionine group.This segment includes a signal sequence of 31 amino acids. The α-chainstarts at amino acid 32 and contains four kringle domains. The so-called“hairpin domain” consists of amino acids 70-96. The kringle 1 domainconsists of amino acids 128-206. The kringle 2 domain consists of aminoacids 211-288, the kringle 3 domain consists of amino acids 305-383, andthe kringle 4 domain consists of amino acids 391-469 of the α-chain,approximately. There exist variations of these sequences, essentiallynot affecting the biological properties of NK4 (especially not affectingits activities antagonistic to HGF and its antiangiogenic activities),which variations are described, for example, in WO 93/23541. Also thelength of NK4 can vary within a few amino acids as long as itsbiological properties are not affected.

[0021] NK4 is composed of the N-terminal 447 amino acids of theHGF/SFα-chain, which includes the above-mentioned hairpin domain and thefour kringle domains. It can be produced recombinantly, either by theproduction of recombinant human HGF/SF and digestion with elastase(Date, K., FEBS Letters 420 (1997) 1-6) or by recombinant expression ofan NK4 encoding nucleic acid in appropriate host cells, as describedbelow. NK4 glycoprotein has a molecular weight of about 57 kDa (52 kDafor the polypeptide part alone) and has the in vivo biological activityof causing inhibition of tumor growth, angiogenesis and/or metastasis.

[0022] “MonoPEGylated NK4” as used herein means that NK4 has attachedcovalently one polyethylene glycol group with a molecular weight of fromabout 20 to about 40 kDa. The group can be attached, preferablyrandomly, at one of various sites of the NK4 molecule, preferably,however, at one of the most reactive sites, e.g., of the lysine sidechains and the N-terminal amino group. MonoPEGylated NK4 (whichtherefore preferably is a mixture of monoPEGylated NK4 molecules,PEGylated at different sites which are the ε-amino groups of NK4 lysineand the N-terminal amino group) is at least 90% of the preparation, andmost preferably, the monoPEGylated NK4 is 92%, or more, of thepreparation of the present invention. The monoPEGylated NK4 preparationsaccording to the invention are homogeneous enough to display theadvantages of a substantially homogeneous preparation, e.g., in apharmaceutical application. “Substantially homogeneous” as used hereinmeans that the only PEG-NK4 conjugate molecules produced, contained orused are those having one PEG group attached. The monoPEGylated NK4preparation of the present invention may contain unreacted (i.e.,lacking PEG group) protein and/or multiPEGylated NK4. As ascertained bypeptide mapping and N-terminal sequencing, one example below provides apreparation which is at least 90% monoPEG-NK4 conjugate and at most 2%unreacted protein.

[0023] The PEG polymer molecules used according to the invention have amolecular weight of from about 20 to about 40 kDa, whereby PEG polymerswith about 20, 30 or 40 kDa are preferred (by “molecular weight” as usedhere there is to be understood the mean molecular weight of the PEG; theterm “about” indicates that in said PEG preparations, some moleculeswill weigh more and some less than the stated molecular weight).

[0024] “PEG or PEG group” according to the invention means a residuecontaining poly(ethylene glycol) as an essential part. Such a PEG cancontain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, EP-A 0 473 084 and U.S. Pat.No. 5,932,462. Especially preferred are PEGs with two PEG side-chains(PEG2) linked via the primary amino groups of a lysine (Monfardini, C.,et al., Bioconjugate Chem. 6 (1995) 62-69). As PEG polymers with amolecular weight of 20-30 kDa linear PEG molecules are preferred and asPEG polymers with a molecular weight of more than 30 kDa, especiallywith 40 kDa, branched PEGs are preferred. As PEG 40 kDa a two-armed PEG(PEG2) is particularly preferred.

[0025] According to the invention a method is provided for theproduction of a monoPEGylated NK4 preparation. PEGylation of NK4 can beperformed according to the methods of the state of the art, for exampleby reaction of NK4 with electrophilically actived PEGs (supplier:Shearwater Corp., USA, www.shearwatercorp.com). Preferred PEG reagentsare, e.g., N-hydroxysuccinimidyl propionates (PEG-SPA) or butanoates(PEG-SBA) or branched N-hydroxysuccinimides such as mPEG2-NHS(Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69). Suchmethods result in an NK4 polypeptide which is randomly PEGylated at anε-amino group of an NK4 lysine or the N-terminal amino group. Notrandomly, N-terminally PEGylated NK4 can be produced according to WO94/01451.

[0026] In a preferred embodiment of the invention, said NK4 iscovalently linked to one poly(ethylene glycol) group of the formula

—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR

[0027] with the —CO (i.e. carbonyl) of the poly(ethylene glycol) groupforming an amide bond with one of the amino groups of NK4; R being loweralkyl; x being 2 or 3; m being from about 450 to about 950; and n and mbeing chosen so that the molecular weight of the conjugate minus the NK4protein is from about 20 to 40 kDa. As amino group of NK4 the ε-aminogroup of NK4 lysine is the available (free) amino group.

[0028] More specifically, the above conjugates may be represented byformula (I)

P—NHCO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR  (I)

[0029] wherein P is the group of an NK4 protein as described herein,(i.e. without the amino group or amino groups which form an amidelinkage with the carbonyl shown in formula (I); and wherein R is loweralkyl; x is 2 or 3; m is from about 450 to about 950 and is chosen sothat the molecular weight of the conjugate minus the NK4 protein is fromabout 20 to about 40 kDa. As used herein, the given ranges of “m” havean orientational meaning. The ranges of “m” are determined in any case,and exactly, by the molecular weight of the PEG group.

[0030] In a further preferred embodiment of the invention, said NK4 iscovalently linked to one poly(ethylene glycol) group of the formula

[0031] wherein y is 1 to 4, preferably 4, n and p together are chosensuch that the molecular weight of the conjugate minus the NK4 protein isfrom about 20 to 40 kDa, preferably 40 kDa, and n and p differ by notmore than 25%, preferably by not more than 10%, and most preferably areidentical, and R is lower alkyl.

[0032] As used herein, “lower alkyl” means a linear or branched alkylgroup having from one to six carbon atoms (C₁-C₆) alkyl). Examples oflower alkyl groups include methyl, ethyl and isopropyl. In accordancewith this invention, R is any lower alkyl. Conjugates in which R ismethyl are preferred.

[0033] The symbol “m” represents the number of ethylene oxide groups(OCH₂CH₂) in the poly(ethylene oxide) group. A single PEG subunit ofethylene oxide has a molecular weight of about 44 daltons. Thus, themolecular weight of the conjugate (excluding the molecular weight of theNK4) depends on the number “m”. In the conjugates of this invention “m”is from about 450 to about 950 (corresponding to a molecular weight ofabout 20 kDa to about 40 kDa). The number m is selected such that theresulting conjugate of this invention has a physiological activitycomparable to unmodified NK4, which activity may represent the same as,more than, or a fraction of the corresponding activity of unmodifiedNK4. A molecular weight of “about” a certain number means that it iswithin a reasonable range of that number as determined by conventionalanalytical techniques. The number “m” is selected so that the molecularweight of each poly(ethylene glycol) group that is covalently linked tothe NK4 protein is from about 20 kDa to about 40 kDa.

[0034] The compound of formula (I) can be prepared, for example, from aknown activated polymeric material:

[0035] in which R and m are as described above, by condensing thecompound of Formula II with the NK4 protein. Compounds of formula (II)in which x is 3 are alpha-lower alkoxybutyric acid succinimidyl estersof poly(ethylene glycol) (lower alkoxy-PEG-SBA). Compounds of formula(II) in which x is 2 are alpha-lower alkoxypropionic acid succinimidylesters of poly(ethylene glycol) (lower alkoxy-PEG-SPA). Any conventionalmethod of reacting an activated ester with an amine to form an amide canbe utilized. In the reaction described above, the exemplifiedsuccinimidyl ester is a leaving group causing the amide formation. Theuse of succinimidyl esters such as the compounds of formula II toproduce conjugates with proteins are disclosed in U.S. Pat. No.5,672,662, issued Sep. 30, 1997 (Harris, et al.).

[0036] Human NK4 contains 30 free ε-amino groups of 30 lysine residues.When the PEGylation reagent was combined with a SBA compound of FormulaII, it has been found that at a protein concentration of about 5 to 10mg/ml, at a pH of about 7.0 to 8.0, a protein:PEG ratio of about 1:3 anda reaction temperature of from 20-25° C., a mixture of mono-, di-, andtrace amounts of the tri-PEGylated species were produced. When theprotein:PEG ratio was about 1:1 or 1:2 (for example, preferably about1:2 for 30 kDa PEG-SBA and about 1:5 for 40 kDa PEG2-NHS), primarily themonoPEGylated species is produced. By manipulating the reactionconditions (e.g., ratio of reagents, pH, temperature, proteinconcentration, time of reaction etc.), the relative amounts of thedifferent monoPEGylated species can be optimized. Typical, but notlimiting, conditions are about 8 to 12 mg/ml NK4, 0.3 M potassiumphosphate, pH 8, 25° C., reaction time of 1 h. Under such conditionsusing 30 kDa PEG-SBA (1:2, protein:PEG), the yield is about 38%monoPEGylated NK4.

[0037] Monopegylated NK4 can also be produced according to the methodsdescribed in WO 94/01451. WO 94/01451 describes a method for preparing arecombinant polypeptide with a modified terminal amino acid alpha-carbonreactive group. The steps of the method involve forming the recombinantpolypeptide and protecting it with one or more biologically addedprotecting groups at the N-terminal alpha-amine and C-terminalalpha-carboxyl. The polypeptide can then be reacted with chemicalprotecting agents to selectively protect reactive side chain groups andthereby prevent side chain groups from being modified. The polypeptideis then cleaved with a cleavage reagent specific for the biologicalprotecting group to form an unprotected terminal amino acid alpha-carbonreactive group. The unprotected terminal amino acid alpha-carbonreactive group is modified with a chemical modifying agent. The sidechain protected terminally modified single copy polypeptide is thendeprotected at the side chain groups to form a terminally modifiedrecombinant single copy polypeptide. The number and sequence of steps inthe method can be varied to achieve selective modification at the N-and/or C-terminal amino acid of the polypeptide.

[0038] Further preferred conjugates according to the invention consistof NK4 protein being covalently linked to a lower-alkoxy poly(ethyleneglycol) group via a linker of the formula

—C(O)—X—S—Y— with the C(O)

[0039] of the linker forming an amide bond with an amino group of NK4(as mentioned above, the ε-amino group of lysine residues is available),X is —(CH₂)_(k)— or —CH₂(O—CH₂—CH₂)_(k)—, k is from 1 to 10, Y is

[0040] the average molecular weight of the poly(ethylene glycol) moietyis from about 20 kDa to about 40 kDa and the molecular weight of theconjugate is from about 72 kDa to about 92 kDa at a molecular weight of52 kDa for NK4 polypeptide, or from about 77 kDa to about 97 kDa at amolecular weight of 57 kDa for NK4 glycoprotein.

[0041] This NK4 species may also be represented by formula (III)

P—NH—CO—X—S—Y—(OCH₂CH₂)_(m)—OR  (III)

[0042] wherein R may be any lower alkyl, by which is meant a linear orbranched alkyl group having from one to six carbon atoms such as methyl,ethyl, isopropyl, etc. A preferred alkyl is methyl. X may be —(CH₂)_(k)—or —CH₂(O—CH₂—CH₂)_(k)—, wherein k is from 1 to about 10. Preferably, kis from 1 to about 4, more preferably, k is 1 or 2. Most preferably, Xis —(CH₂).

[0043] In formula III, Y is

[0044] In formula (III), the number m is selected such that theresulting conjugate of formula (III) has a physiological activitycomparable to unmodified NK4, which activity may represent the same as,more than, or a fraction of the corresponding activity of unmodifiedNK4. m represents the number of ethylene oxide chains in the PEG unit. Asingle PEG subunit of —(OCH₂CH₂)— has a molecular weight of about 44daltons. Thus, the molecular weight of the conjugate (excluding themolecular weight of the NK4) depends on the number m. A molecular weightof “about” a certain number means that it is within a reasonable rangeof that number as determined by conventional analytical techniques. m istherefore an integer ranging from about 450 to about 950 (correspondingto a molecular weight of from about 20 to about 40 kDA).

[0045] Preferred NK4 proteins of formula (III) are represented by theformulae:

[0046] Most preferred NK4 protein products are represented by theformula:

[0047] These NK4 proteins may be prepared by

[0048] (a) covalently reacting a free amino group, preferably an ε-aminogroup of a lysine amino acid of an NK4 protein or the N-terminal aminogroup represented by the formula, P-NH₂, with a bi-functional reagentrepresented by the formula, Z—CO—X—S—Q, to form an intermediate with anamide linkage represented by the formula:

P—NH—CO—X—S—Q

[0049] wherein P is an NK4 protein less the amino group which forms anamide linkage; Z is a reactive group, e.g. a carboxylic-NHS ester; X is—(CH₂)k— or —CH₂(O—CH₂—CH₂)_(k)—, wherein k is from 1 to about 10; and Qis a protecting group, like alkanoyl, e.g. acetyl.

[0050] (b) covalently reacting the intermediate with an amide linkagefrom step (a) with an activated polyethylene glycol derivativerepresented by the formula, W—[OCH₂CH₂]_(m)—OR, to form an NK4 proteinproduct represented by the formula:

[0051] wherein W is a sulfhydryl reactive form of Y; m is an integerranging from about 450 to about 950; R is lower alkyl; and Y is asdefined above.

[0052] In this embodiment, the bi-functional reagent is preferablyN-succinimidyl-S-acetylthiopropionate orN-succinimidyl-S-acetylthioacetate, Z is preferablyN-hydroxy-succinimide, and the activated polyethylene glycol derivativeW—[OCH₂CH₂]_(m)—OR is preferably selected from the group consisting ofiodo-acetyl-methoxy-PEG, methoxy-PEG-vinylsulfone, andmethoxy-PEG-maleimide.

[0053] In more detail, the NK4 proteins of formula (III) may be preparedby covalent linking of a thiol group to NK4 (“activation”) and couplingthe resulting activated NK4 with a poly(ethylene glycol) (PEG)derivative. The first step for the preparation of monoPEGylated NK4according to the present invention comprises covalent linking of a thiolgroup via NH₂-groups of NK4. This activation of NK4 is performed withbi-functional reagents which carry a protected thiol group and anadditional reactive group, such as active esters (e.g., asuccinimidylester), anhydrides, esters of sulphonic acids, halogenidesof carboxylic acids and sulphonic acids, respectively. The thiol groupis protected by groups known in the art, e.g., acetyl groups. Thesebi-functional reagents are able to react with the amino groups byforming an amide linkage.

[0054] In a preferred embodiment the activation of the amino groups isperformed by reaction with bi-functional reagents having a succinimidylmoiety. The bi-functional reagents may carry different spacer species,e.g. —(CH₂)_(k)— or —CH₂—(O—CH₂—CH₂—)_(k)— moieties, wherein k is from 1to about 10, preferably from 1 to about 4, and more preferably 1 or 2,and most preferably 1. Examples of these reagents areN-succinimidyl-S-acetylthiopropionate (SATP) andN-succinimidyl-S-acetylthioacetate (SATA)

[0055] Acetylthioalkyl-carboxylic-NHS-ester, like

[0056] 2-(Acetylthio)-(ethoxy)_(k)-acetic-acid-NHS-ester

[0057] with k as defined above.

[0058] The preparation of the bi-functional reagents is known in theart. Precursors of 2-(acetylthio)-(ethoxy)_(k)-acetic-acid-NHS-estersare described in DE-3924705, while the derivatization to the acetylthiocompound is described by March, J., Advanced Organic Chemistry (1977)375-376. SATA is commercially available (Molecular Probes, Eugene,Oreg., USA and Pierce, Rockford, Ill.).

[0059] The addition of only one thiol group to an NK4 molecule can beselected by adjusting the reaction parameters, i.e., the protein (NK4)concentration and the protein/bi-functional reagent ratio.

[0060] The reaction is carried out, for example, in an aqueous buffersolution, pH 6.5-8.0, e.g., in 10 or 100 mM potassium phosphate, with orwithout 300 mM NaCl, pH 7.3. The bi-functional reagent may be added inDMSO. After completion of the reaction, preferably after 30 minutes, thereaction is stopped by addition of lysine. Excess bifunctional reagentmay be separated by methods known in the art, e.g., by dialysis orcolumn filtration. The average number of thiol groups added to NK4 canbe determined by photometric methods described in, for example,Grasetti, D. R., and Murray, J. F. in J. Appl. Biochem. Biotechnol. 119(1967) 41-49.

[0061] The above reaction is followed by covalent coupling of anactivated polyethylene glycol (PEG) derivative. Suitable PEG derivativesare activated PEG molecules with an average molecular weight of fromabout 20 to about 40 kDa.

[0062] Activated PEG derivatives are known in the art and are describedin, for example, Morpurgo, M., et al. J. Bioconj. Chem. 7 (1996) 363 fffor PEG-vinylsulfone. Linear chain and branched chain PEG species aresuitable for the preparation of the compounds of Formula 1. Examples ofreactive PEG reagents are iodo-acetyl-methoxy-PEG andmethoxy-PEG-vinylsulfone:

[0063] The use of these iodo-activated substances is known in the artand described e.g. by Hermanson, G. T., in Bioconjugate Techniques,Academic Press, San Diego (1996) p. 147-148.

[0064] Most preferably, the PEG species are activated by maleimide using(alkoxy-PEG-maleimide), such as methoxy-PEG-maleimide (MW 20,000 to40,000; Shearwater Polymers, Inc.). The structure ofalkoxy-PEG-maleimide is as follows:

[0065] with R and m are as defined above, preferably

[0066] The coupling reaction with alkoxy-PEG-maleimide takes place afterin situ cleavage of the thiol protecting group in an aqueous buffersolution, e.g. 10 mM potassium phosphate, 300 mM NaCl, 2 mM EDTA, pH6.2. The cleavage of the protecting group may be performed, for example,with hydroxylamine in DMSO at 25° C., pH 6.2 for about 90 minutes. Forthe PEG modification the molar ratio of activatedNK4/alkoxy-PEG-maleimide should be from about 1:1 to about 1:6. Thereaction may be stopped by addition of cysteine and reaction of theremaining thiol (—SH) groups with N-methylmaleimide or other appropriatecompounds capable of forming disulfide bonds. Because of the reaction ofany remaining active thiol groups with a protecting group such asN-methylmaleimide or other suitable protecting group, the NK4 proteinsin the conjugates of this invention may contain such protecting groups.Generally the procedure described herein will produce a mixture ofmolecules having varying numbers of thiols protected by differentnumbers of the protecting group, depending on the number of activatedthiol groups on the protein that were not conjugated to PEG-maleimide.

[0067] Whereas N-methylmaleimide forms the same type of covalent bondwhen used to block the remaining thiol-groups on the PEGylated protein,disulfide compounds will lead in an intermolecular sulfide/disulfideexchange reaction to a disulfide bridged coupling of the blockingreagent. Preferred blocking reagents for that type of blocking reactionare oxidized glutathione (GSSG), cysteine and cystamine. Whereas withcysteine no additional net charge is introduced into the PEGylatedprotein, the use of the blocking reagents GSSG or cystamine results inan additional negative or positive charge.

[0068] The further purification of the compounds of formula (III),including the separation of mono- from di-, tri- and multi-PEGylated NK4species, may be done by methods known in the art, e.g., columnchromatography. The percentage of mono-PEG conjugates can be controlledby pooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety percent mono-PEGconjugates is a good balance of yield and activity. Compositions inwhich, for example, at least ninety-two percent or at least ninety-sixpercent of the conjugates are mono-PEG species may be desired. In anembodiment of this invention the percentage of mono-PEG conjugates isfrom ninety percent to ninety-six percent.

[0069] The compounds of the present invention can be formulatedaccording to methods for the preparation of pharmaceutical compositionswhich methods are known to the person skilled in the art. For theproduction of such compositions, monoPEGylated NK4 according to theinvention is combined in a mixture with a pharmaceutically acceptablecarrier. Such acceptable carriers are described, for example, inRemington's Pharmaceutical Sciences, 18^(th) edition, 1990, MackPublishing Company, edited by Oslo et al. (e.g. pp. 1435-1712). Typicalcompositions contain an effective amount of the substance according tothe invention, for example from about 0.1 to 100 mg/ml, together with asuitable amount of a carrier. The compositions may be administeredparenterally.

[0070] This invention further provides pharmaceutical compositionscomprising monoPEGylated NK4, in which the percentage of mono-PEGconjugates is preferably at least ninety percent, more preferably atleast ninety-two percent.

[0071] The pharmaceutical compositions according to the invention can beprepared according to known methods in the art. Usually, solutions ofmonoPEGylated NK4 are dialyzed against the buffer intended to be used inthe pharmaceutical composition and the desired final proteinconcentration is adjusted by concentration or dilution.

[0072] Such pharmaceutical compositions may be used for administrationfor injection and contain an effective amount of the monoPEGylated NK4together with pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents of various buffer contents (e.g. arginine, acetate,phosphate), pH and ionic strength, additives such as detergents andsolubilizing agents (e.g. Tween 80/polysorbate, pluronic F68, sodiumchloride, sodium sulfate), antioxidants (e.g. ascorbic acid,L-methionine), preservatives (Timersol, benzyl alcohol) and bulkingsubstances (e.g. saccharose, mannitol), incorporation of the materialinto particulate preparations of polymeric compounds such as polylacticacid, polyglycolic acid, etc. or into liposomes. Such compositions mayinfluence the physical state stability rate of release and clearance ofthe monoPEGylated NK4 according to the invention.

[0073] The present invention provides for improved NK4 activity and apharmaceutical composition which can be administered in only a few bolusapplications per week and/or in very low dosages, and which are capableof suppressing tumor growth, angiogenesis and metastasis.

[0074] It was surprisingly found that the entire amount of randomlyPEGylated NK4 according to the invention to be administered duringtreatment is considerably lower compared to administering unPEGylatedNK4. Therefore, the amount of PEGylated NK4 used in pharmaceuticaltreatment is about 50% or lower, preferably about 20% or lower, and mostpreferably about 10% or lower than the required amount of unPEGylatedNK4.

[0075] The invention provides methods for the treatment of human cancer(e.g. breast, lung, prostate, pancreas or colon cancer) characterized inthat a pharmaceutically effective amount of monoPEGylated NK4 isadministered in one to seven bolus applications per week to the patientin need thereof.

[0076] Typically, in a standard cancer treatment regimen, patients aretreated with dosages in the range between from 1 to 30 mg ofmonoPEGylated NK4 per kg per day over a certain period of time, lastingfrom one day to about 30 days or even longer. Drug is applied as asingle daily subcutaneous or i.v. bolus injection of a pharmaceuticalformulation containing 0.1 to 100 mg monoPEGylated NK4 per ml. Thistreatment can be combined with any standard (e.g. chemotherapeutic)treatment, by applying monoPEGylated NK4 before, during or after thestandard treatment. This results in an improved outcome compared tostandard treatment alone.

[0077] In any case the overall amount of administered PEGylated NK4according to the invention is considerably lower than the amount of NK4for the same treatment.

[0078] The following examples, references and the sequence listing areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

EXAMPLE 1 Recombinant Production of NK4

[0079] NK4 for therapeutic uses may be produced by recombinant meansusing bacterial or eukaryotic expression systems. Suitable eukaryoticexpression systems are for example engineered HeLa, BHK or preferablyCHO cells. Cells engineered for NK4 production are cultivated in asuitable medium. Typically, a 1 to 5 liter cell culture is used asinoculum for a 10 liter fermenter. After 3 to 5 days, the culture in the10 liter fermenter can be used as inoculum for the 100 liter fermenter.After additional 3 to 5 days of fermentation, this culture can be usedas inoculum for the 1000 liter production fermenter. After 3 to 4 dayscells are removed by filtration or centrifugation and discarded. The NK4containing supernatant is filtered, collected and processed duringpurification. The purification process is described in the followingexample.

EXAMPLE 2 Purification

[0080] Heparin-Sepharose consists of Separose beads to the surface ofwhich heparin is covalently bound. Since NK4 shows a high affinity toheparin it is retained on this column and can be eluted with high saltconcentrations, whereas protein contaminants and other impurities eitherdo not bind or elute at lower salt concentrations. NK4 containingfractions, eluting at about 0.7 to 1.1 M NaCl in 50 mM Hepes pH 7.5 arecollected and loaded onto a hydroxyapatite column. NK4 elutes with about0.4 to 0.7 M potassium phosphate, pH 7.5. The resulting fractions aresubstantially free of contaminating proteins and can be further purifiedby S-sepharose chromatography.

EXAMPLE 3 Production of MonoPEGylated NK4

[0081] NK4 purified in accordance with the above mentioned procedure wasused for PEGylation reactions. Three suitable methods are described.

[0082] a) PEGylation of NK4 with mPEG-SBA

[0083] Aliquots of NK4 were reacted with methoxy-PEG-SBA (5 kDa forcomparison, 20 kDa and 30 kDa, respectively; Shearwater Polymers, Inc.,Huntsville Ala.). Reaction was carried out at a protein to reagent ratiobetween 1:1 and 1:5 for about 2 h at room temperature (a ratio of 1:2 ispreferred when using 20 and 30 kDa PEG). The reaction was stopped by theaddition of 10 mM Tris-buffer or arginine HCl, pH 8, and samples wereanalyzed by SDS-PAGE or size exclusion chromatography on a Superose 6column (Pharmacia) using as buffer solution 500 mmol/l potassiumphosphate, pH 6.8, for equilibration and elution. The reaction wasoptimized by varying protein to reagent ratio, pH, time and temperature,in order to obtain predominantly monoPEGylated NK4.

[0084] Such conditions are, for example:

[0085] Concentration NK4: 8-12 mg/ml

[0086] Buffer system/pH: 0.3 M potassium phosphate, pH 8

[0087] Temperature: 25° C.

[0088] Reaction time: 1 h

[0089] Molar ratios (protein:reagent): 1:2

[0090] Yield:

[0091] MonoPEGylated NK4: 38%

[0092] DiPEGylated NK4: 17%

[0093] UnPEGylated NK4: 45%

[0094] b) PEGylation of NK4 with mPEG-SPA

[0095] Aliquots of NK4 (protein concentration 8 to 12 mg/ml in 0.3 Mpotassium phosphate, pH 8) were reacted with methoxy-PEG-SPA (5 kDa forcomparison and 20 kDa, respectively; Shearwater Polymers, Inc.,Huntsville Ala.). Reaction was carried out at a protein to reagent ratioof 1:2 for about 2 h at room temperature. The reaction was stopped bythe addition of 10 mM Tris-buffer or arginine HCl and samples wereanalyzed by SDS-PAGE, reversed phase HPLC or size exclusionchromatography on a Superose 6 column (Pharmacia) using as buffersolution 500 mmol/l potassium phosphate, pH 6.8, for equilibration andelution. The reaction was optimized by varying protein to reagent ratio,pH, time and temperature, in order to obtain predominantly monoPEGylatedNK4, compared to di-and tri-PEGylated NK4.

[0096] c) PEGylation of NK4 with mPEG2-NHS

[0097] This PEGylation was performed as described in Example 3b with theexception that instead of PEG-SPA, mPEG2-NHS (40 kDa PEG, branched via alysine linker) was used at a molar ratio of 1:5 (protein:PEG reagent).

EXAMPLE 4 MonoPEGylated

[0098] MonoPEGylated NK4 can be separated from unPEGylated, di- andtri-PEGylated NK4 by running a preparative size exclusion chromatography(e.g. Superose 6 or Superdex 200; Pharmacia) using as buffer solution500 mmol/l K-phosphate pH 6.8, for equilibration and elution, or by ionexchange chromatography. The purified protein contains predominantly themonoPEGylated species. Fractions were collected and analyzed by SDS-PAGEand reversed phase chromatography.

EXAMPLE 5 Molecular Characterization of MonoPEGylated NK4

[0099] a) Size Exclusion Chromatography

[0100] The monoPEGylated species elutes earlier in size exclusionchromatography (e.g. Superose 6 or Superdex 200; Pharmacia; using asbuffer solution 500 mmol/l K-phosphate pH 6.8, for equilibration andelution) as compared to the unmodified form. This is due to an increasedhydrodynamic radius of the molecule.

[0101] b) SDS-PAGE

[0102] In SDS-PAGE proteins are separated according to their molecularweight. Due to an increase in molecular weight by PEGylation, themonoPEGylated NK4 shows a shorter migration distance as compared to theunmodified NK4. The migration distance is inversely correlated with thechain length of the PEG moiety and the number of PEG groups attached perNK4 molecule.

[0103] c) Peptide Mapping

[0104] Digestion of monoPEGylated NK4 with sequence-specificendo-proteinases (e.g. LysC or trypsin) results in a characteristicpeptide map. The resulting peptides can be separated by reversed phasechromatography and analyzed by mass spectrometry and/or N-terminalsequencing. This allows for a determination of the PEG-modified groupswithin the NK4 molecule.

[0105] d) Reversed Phase Chromatography

[0106] MonoPEGylated NK4 can also be characterized by reversed phasechromatography. PEGylation of NK4 results in a change in retention timeas compared to unmodified NK4.

EXAMPLE 6 Comparison of MonoPEGylated, UnPEGylated and Multi-PEGylatedNK4

[0107] In this example, unPEGylated NK4, NK4 monoPEGylated with PEG 5kDa, PEG 20 kDa, PEG 30 kDa, PEG 40 kDa and multiPEGylated NK4 (NK4PEGylated with more than one PEG chain ) were used.

[0108] a) Scatter Assay

[0109] MDCK cells were subconfluently grown in tissue culture plates.Cells were treated with HGF (10 ng/ml) or with combinations of HGF andNK4. In these experiments the HGF-induced cell scattering was inhibitedby the addition of a 10 to 1000-fold molar excess of NK4, showing thefunctional activity of PEGylated NK4. It was found that the in vitroactivity of monoPEGylated 5 kDa-PEG-NK4, 20 kDa-PEG-NK4, 30 kDa-PEG-NK4,and 40 kDa-PEG-NK4 is similar to unPEGylated NK4. It was also found thatthe addition of more than one PEG chain (20 to 40 kDa) results in asignificant loss of in vitro activity (Table 1). TABLE 1 Scores of theMDCK scatter assay NK4 (control) 40kDa-PEG-NK4 30kDa-PEG-NK4 NK4 Mono-Di- Tri- Mono- Di- and Tri- Tri- and Penta- (μg/ml) NK4 NK4 PEG PEG PEGPEG PEG Tetra-PEG PEG 5.00 ++ ++ ++ + +/− ++ ++ + − 1.67 ++ ++ ++ + −++ + +/− − 0.56 ++ ++ ++ +/− − ++ + − − 0.19 + + + − − + +/− − − 0.06+/− +/− +/− − − +/− − − − 0.02 − − − − − − − − − 20kDa-PEG-NK45kDa-PEG-NK4 NK4 Mono- Di- Tri- Mono- Di- Tri- Tetra- Penta- Hexa-Hepta- (μg/ml) PEG PEG PEG PEG PEG PEG PEG PEG PEG PEG 5.00 ++ ++ + ++++ ++ ++ + − − 1.67 ++ + + ++ ++ ++ ++ + − − 0.56 ++ + +/− ++ ++ + + +/−− − 0.19 + +/− − + + + +/− − − − 0.06 +/− − − +/− +/− − − − − − 0.02 − −− − − − − − − −

[0110] The relative potency of various PEGylated forms of NK4 in theinhibition of HGF (10 ng/ml)-induced scattering of MDCK cells wasassayed. ++ means complete inhibition, + means inhibition, +/− meansweak inhibition and − means no inhibition.

[0111] b) HUVEC Proliferation Assay

[0112] Inhibition of the mitogenic activity of HGF by NK4 was determinedby measuring proliferation of HUVECs in culture as described inNakamura, T., et al., Nature 342 (1989) 440-443. In these experimentsthe HGF-induced cell proliferation was inhibited by the addition of a 10to 1000-fold molar excess of NK4, showing the functional activity ofmonoPEGylated NK4 (FIGS. 1 and 2).

[0113] Alternatively, the HUVEC Proliferation assay was performed byinhibition of the mitogenic activity of bFGF (basic fibroblastic growthfactor) by NK4. Inhibition was determined by measuring proliferation ofHUVECs in culture as described in Kuba, K., et al., Cancer Res. 60(2000) 6737-6743. In these experiments the FGF-induced cellproliferation was inhibited by the addition of a 10 to 1000-fold molarexcess of NK4, showing the functional activity of monoPEGylated NK4(FIG. 3).

[0114] c) Invasion Assay

[0115] In this assay the invasive potential of tumor cells is analyzed.The assay was done essentially as decribed in Albini, A., et al. (1987)using HT115 cells. Again, HGF-induced (10 ng/ml) cell invasion could beinhibited by a 10 to 1000-fold molar excess of PEGylated NK4, showingthe functional activity of monoPEGylated NK4.

EXAMPLE 7 Activity in vivo

[0116] Model: Panc Tul human pancreatic cancer orthotopic SCID mousemodel (Alves, F., et al., Pancreas 23 (2001) 227-235).

[0117] Treatment: After 8 days, one application daily of monoPEGylatedNK4 over a period of 21 days.

[0118] Doses:

[0119] 16 mg/kg/day

[0120] 4 mg/kg/day

[0121] 1 mg/kg/day

[0122] Placebo

[0123] Result: Treatment with monoPEGylated NK4 shows a dose-dependentsuppression of primary tumor growth compared to placebo treated groups.For example, with a dose of 4 mg/kg/day of monoPEGylated NK4, a tumorvolume of only 200 mm³ was found compared to a tumor volume of 280 mm³in the placebo treated animal group.

EXAMPLE 8 Pharmaceutical Composition

[0124] Suitable pharmaceutical compositions are, for example:

[0125] 1 to 30 mg/ml monoPEGylated NK4

[0126] 150 mM NaCl

[0127] 10 mM sodium phosphate, pH 7.2

[0128] 1 to 30 mg/ml monoPEGylated NK4

[0129] 150 mM NaCl

[0130] 0.01% Tween 80 or Tween 20 or pluronic F68

[0131] 10 mM sodium phosphate, pH 7.2

[0132] 1 to 30 mg/ml monoPEGylated NK4

[0133] 50 mM NaCl

[0134] 3% mannitol

[0135] 10 mM sodium phosphate, pH 7.2

[0136] 1 to 30 mg/mil monoPEGylated NK4

[0137] 50 mM NaCl

[0138] 3% mannitol

[0139] 0.01% Tween 80 or Tween 20 or pluronic F68

[0140] 10 mM sodium phosphate, pH 7.2

[0141] The compositions are prepared in that monoPEGylated NK4 isdialyzed against the above mentioned buffer solution (with or withoutmannitol). The protein concentration is adjusted by concentration ordilution with the buffer solution. Detergent and NaCl are added out of a10% stock solution.

EXAMPLE 9 Pharmacokinetic Analysis of NK4, 30 kDa-mono-PEG-NK4 and 40kDa-mono-PEG-NK4

[0142] Adult mice (4 per group) received single i.v. or s.c. bolusinjections of NK4, 30 kDa-mono-PEG-NK4 or 40 kDa-mono-PEG-NK4 (4 mg/kgin 0.25 ml injection volume), respectively. At several time points bloodsamples were taken and analyzed for NK4, 30 kDa-mono-PEG-NK4 or 40kDa-mono-PEG-NK4 content by ELISA. The time-concentration curves werecalculated and are shown in FIGS. 4A and 4B. These data show that 30kDa-mono-PEG-NK4 and 40 kDa-mono-PEG-NK4 have significantly improvedstability in vivo, resulting in significantly increased plasmahalf-lives compared to unmodified NK4.

1 2 1 1344 DNA homo sapiens 1 caaaggaaaa gaagaaatac aattcatgaattcaaaaaat cagcaaagac taccctaatc 60 aaaatagatc cagcactgaa gataaaaaccaaaaaagtga atactgcaga ccaatgtgct 120 aatagatgta ctaggaataa aggacttccattcacttgca aggcttttgt ttttgataaa 180 gcaagaaaac aatgcctctg gttccccttcaatagcatgt caagtggagt gaaaaaagaa 240 tttggccatg aatttgacct ctatgaaaacaaagactaca ttagaaactg catcattggt 300 aaaggacgca gctacaaggg aacagtatctatcactaaga gtggcatcaa atgtcagccc 360 tggagttcca tgataccaca cgaacacagctttttgcctt cgagctatcg gggtaaagac 420 ctacaggaaa actactgtcg aaatcctcgaggggaagaag ggggaccctg gtgtttcaca 480 agcaatccag aggtacgcta cgaagtctgtgacattcctc agtgttcaga agttgaatgc 540 atgacctgca atggggagag ttatcgaggtctcatggatc atacagaatc aggcaagatt 600 tgtcagcgct gggatcatca gacaccacaccggcacaaat tcttgcctga aagatatccc 660 gacaagggct ttgatgataa ttattgccgcaatcccgatg gccagccgag gccatggtgc 720 tatactcttg accctcacac ccgctgggagtactgtgcaa ttaaaacatg cgctgacaat 780 actatgaatg acactgatgt tcctttggaaacaactgaat gcatccaagg tcaaggagaa 840 ggctacaggg gcactgtcaa taccatttggaatggaattc catgtcagcg ttgggattct 900 cagtatcctc acgagcatga catgactcctgaaaatttca agtgcaagga cctacgagaa 960 aattactgcc gaaatccaga tgggtctgaatcaccctggt gttttaccac tgatccaaac 1020 atccgagttg gctactgctc ccaaattccaaactgtgata tgtcacatgg acaagattgt 1080 tatcgtggga atggcaaaaa ttatatgggcaacttatccc aaacaagatc tggactaaca 1140 tgttcaatgt gggacaagaa catggaagacttacatcgtc atatcttctg ggaaccagat 1200 gcaagtaagc tgaatgagaa ttactgccgaaatccagatg atgatgctca tggaccctgg 1260 tgctacacgg gaaatccact cattccttgggattattgcc ctatttctcg ttgtgaaggt 1320 gataccacac ctacaatagt ctaa 1344 2447 PRT homosapiens 2 Gln Arg Lys Arg Arg Asn Thr Ile His Glu Phe LysLys Ser Ala Lys 1 5 10 15 Thr Thr Leu Ile Lys Ile Asp Pro Ala Leu LysIle Lys Thr Lys Lys 20 25 30 Val Asn Thr Ala Asp Gln Cys Ala Asn Arg CysThr Arg Asn Lys Gly 35 40 45 Leu Pro Phe Thr Cys Lys Ala Phe Val Phe AspLys Ala Arg Lys Gln 50 55 60 Cys Leu Trp Phe Pro Phe Asn Ser Met Ser SerGly Val Lys Lys Glu 65 70 75 80 Phe Gly His Glu Phe Asp Leu Tyr Glu AsnLys Asp Tyr Ile Arg Asn 85 90 95 Cys Ile Ile Gly Lys Gly Arg Ser Tyr LysGly Thr Val Ser Ile Thr 100 105 110 Lys Ser Gly Ile Lys Cys Gln Pro TrpSer Ser Met Ile Pro His Glu 115 120 125 His Ser Phe Leu Pro Ser Ser TyrArg Gly Lys Asp Leu Gln Glu Asn 130 135 140 Tyr Cys Arg Asn Pro Arg GlyGlu Glu Gly Gly Pro Trp Cys Phe Thr 145 150 155 160 Ser Asn Pro Glu ValArg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser 165 170 175 Glu Val Glu CysMet Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met 180 185 190 Asp His ThrGlu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr 195 200 205 Pro HisArg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe 210 215 220 AspAsp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys 225 230 235240 Tyr Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr 245250 255 Cys Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr260 265 270 Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val AsnThr 275 280 285 Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln TyrPro His 290 295 300 Glu His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys AspLeu Arg Glu 305 310 315 320 Asn Tyr Cys Arg Asn Pro Asp Gly Ser Glu SerPro Trp Cys Phe Thr 325 330 335 Thr Asp Pro Asn Ile Arg Val Gly Tyr CysSer Gln Ile Pro Asn Cys 340 345 350 Asp Met Ser His Gly Gln Asp Cys TyrArg Gly Asn Gly Lys Asn Tyr 355 360 365 Met Gly Asn Leu Ser Gln Thr ArgSer Gly Leu Thr Cys Ser Met Trp 370 375 380 Asp Lys Asn Met Glu Asp LeuHis Arg His Ile Phe Trp Glu Pro Asp 385 390 395 400 Ala Ser Lys Leu AsnGlu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala 405 410 415 His Gly Pro TrpCys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr 420 425 430 Cys Pro IleSer Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val 435 440 445

What is claimed is:
 1. A conjugate consisting of a NK4 molecule and apolyethylene glycol group having a molecular weight of from about 20 toabout 40 kDa.
 2. The conjugate according to claim 1, wherein saidpolyethylene glycol group has the formula —CO—(CH₂)_(X)—(OCH₂CH₂)_(m)ORand said —CO group forms an amide bond with one of the amino groups ofthe N-terminal fragment of said NK4, wherein X is 2 or 3; m is fromabout 450 to about 950; R is (C₁-C₆)alkyl.
 3. The conjugate according toclaim 1, wherein said polyethylene glycol group has the formula

and said —CO group forms an amide bond with one of the amino groups ofthe N-terminal fragment of said NK4 molecule, wherein y is from 1 to 10;n and p together are from about 450 to about 950; and R is (C₁-C₆)alkyl.4. The conjugate according to claim 1, wherein the polyethylene glycolgroup has a molecular weight of from about 30 to about 40 kDa.
 5. Theconjugate according to claim 1, wherein said polyethylene glycol groupis selected from monomethoxy polyethylene glycol groups.
 6. Theconjugate according to claim 1, wherein the polyethylene glycol group isselected from the group of linear PEG chains and branched PEG chains. 7.The conjugate according to claim 6, wherein the branched PEG chainconsists of two PEG chains and said branched PEG chain is attached tothe NK4 molecule by a primary amino group of lysine of the NK4 molecule.8. The conjugate according to claim 1, wherein the polyethylene glycolgroup is attached to a group selected from the lysine side chains andthe N-terminal amino group of the NK4 molecule.
 9. A method for thetreatment of cancer, comprising administering to a patient in needthereof a pharmaceutical composition comprising conjugates of NK4molecules monoPEGylated with polyethylene glycol groups that have amolecular weight of from about 20 to about 40 kDa, wherein saidconjugates are administered in an amount of from 1 to 30 mgmonoPEGylated NK4 per kg per day.
 10. The method according to claim 9,wherein said monoPEGylated conjugates comprise at least 90% of the totalof pegylated NK4 molecules and unpegylated NK4 molecules in thepharmaceutical composition.
 11. The method according to claim 9, whereinsaid monoPEGylated conjugates comprise conjugates in which thepolyethylene glycol groups are attached to more than one group selectedfrom the lysine side chains of NK4 molecules and conjugates in which thepolyethylene glycol groups are attached to the N-terminal amino groupsof NK4 molecules.
 12. A pharmaceutical composition comprising conjugatesof claim 1 and at least one pharmaceutically acceptable carrier.
 13. Acomposition comprising conjugates of NK4 monoPEGylated with polyethyleneglycol groups that have a molecular weight of from about 20 to about 40kDa, wherein the conjugates comprise conjugates in which thepolyethylene glycol groups are attached to groups selected from thelysine side chains of NK4 molecules and conjugates in which thepolyethylene glycol groups are attached to the N-terminal amino groupsof NK4 molecules.
 14. A composition comprising conjugates of NK4monoPEGylated with polyethylene glycol groups that have a molecularweight of from about 20 to about 40 KDa, wherein said monoPEGylatedconjugates comprise at least 90% of the total of pegylated NK4 moleculesand unpegylated NK4 molecules in the composition.
 15. The compositionaccording to claim 14, wherein said monoPEGylated conjugates comprise atleast 92% of the total of pegylated NK4 molecules and unpegylated NK4molecules in the composition.